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Previous Article | Next Article 
Molecular and Cellular Biology, November 2007, p. 7439-7450, Vol. 27, No. 21
0270-7306/07/$08.00+0 doi:10.1128/MCB.00963-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Marco Saponaro,1,2
Anastasia Baryshnikova,1,2,
Jeong-Hoon Kim,3
Yeon-Soo Seo,3 and
Giordano Liberi1,2*
FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy,1 Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy,2 Department of Biological Sciences, National Creative Research Initiative Center for Cell Cycle Control, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea3
Received 31 May 2007/ Returned for modification 28 June 2007/ Accepted 14 August 2007
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ABSTRACT |
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INTRODUCTION |
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The Saccharomyces cerevisiae Srs2 protein is a 3'-5' DNA helicase (40) structurally and functionally related to bacterial UvrD (2, 51). It is thought that Srs2 plays a key role in influencing the choice between homologous recombination (HR) and postreplication repair (PRR) pathways, both of which are required to counteract the accumulation of gaps during DNA replication (5, 45). A body of evidence suggests that Srs2 inhibits HR at an early step (1, 7, 11, 20, 29, 39, 44), acting as a DNA translocase that disassembles the Rad51 nucleofilament (26, 52). Accordingly, srs2
mutants show hyperactivation of spontaneous recombination events (41), and unrestrained HR is the source of cell death in srs2 mutants when other factors operating at later stages in recombination are also inactivated. This is the case for Sgs1 (14) and Rad54 (23) helicases, which are involved in the resolution of mature recombination intermediates and in promoting D-loop formation and/or stabilization (47, 55), respectively.
Current models indicate that Srs2 inhibits HR and channels DNA lesions towards the PRR pathway. This model is mainly supported by the observation that the DNA damage sensitivity of PRR mutants can be rescued by SRS2 inactivation in the presence of a functional HR pathway (5, 45). The PRR pathway seems to be required to tolerate rather than immediately repair the DNA damage, using both specialized translesion synthesis DNA polymerases and a less-characterized error-free repair branch that is thought to involve a recombination-dependent replication mechanism, such as template switching (5). The PRR pathway is promoted by two diverse protein complexes that contain ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes and that modify PCNA on lysine (K) 164. In particular, the Rad6 (E2)-Rad18 (E3) complex mono-ubiquitylates PCNA on K164, while Rad5 (E3), along with the heterodimeric Mms2-Ubc13 (E2 variant) enzyme, subsequently attaches polyubiquitin chains via K63 to the mono-ubiquitylated K164 residue (18). Polyubiquitin chains linked by K63 isopeptide bonds, in contrast to those assembled in K48 conformation, do not mark the modified proteins for degradation but, rather, signal for other cellular transactions (37). The K164 residue of PCNA can also be sumoylated by a Ubc9/Siz1-dependent pathway (18). Recent evidence obtained with budding yeast indicates that the modification status of PCNA is crucial in determining the PRR subpathway that will be engaged in front of a lesion. In particular, the Srs2 antirecombinogenic function is enhanced by its physical interaction with sumoylated PCNA (34, 36), sumoylation and mono-ubiquitylation of PCNA contribute to spontaneous mutagenesis mediated by translesion DNA polymerases (46), and the polyubiquitylation of PCNA might promote the error-free PRR subpathway (18). In spite of the suggestion that sumoylated PCNA, by recruiting Srs2 at replication forks, would play a key role in preventing HR during S phase (34, 36), recent studies failed to detect the accumulation of recombination intermediates at damaged replication forks in srs2 or even PCNA mutants, in which sumoylation and/or ubiquitylation is abrogated (4, 29). On the other hand, recombination intermediates broadly accumulate at damaged forks in sgs1 or ubc9/mms21 mutants (4, 29), suggesting that the control of HR during replication is far more complicated than the sole regulation of Srs2. Moreover, a number of srs2 recombination phenotypes cannot easily be explained by considering Srs2 only as a protein that counteracts Rad51-mediated strand invasion. Rather, many genetic findings suggest that Srs2 operates at diverse levels in recombination, downstream of the strand invasion step (39), and possibly in promoting double-strand-break (DSB) repair by HR (3, 19, 20, 35).
Despite its key role in maintaining genome integrity in yeast, Srs2 has no human orthologues identified so far. However, the Srs2 DNA helicase has been conserved in other fungi, such as Schizosaccharomyces pombe and Neurospora crassa, and the genetic characterization of null mutants in both organisms showed that the Srs2 orthologues are fundamental players in HR (10, 30, 31, 48, 53). Genetic analysis carried out with fission yeast suggests that Srs2 has overlapping functions in processing recombination intermediates with both Rqh1 and Fbh1 DNA helicases (32, 33). Fbh1 orthologues have been found in mice, chickens, and humans but not in budding yeast (21, 24). In DT40 cells, Fbh1 and BLM proteins cooperate in constraining the extent of sister chromatid exchanges (SCEs) in HR (24). Unusually for DNA unwinding enzymes, Fbh1 DNA helicases possess an F-box motif (22). Studies performed on human cell extracts indicated that human FBH1 (hFBH1), through the F-box domain, is part of a Skp-Cullin-F-box (SCF) ubiquitin ligase complex; while it has been shown that the E3 SCF complex containing hFBH1 promotes the assembly of polyubiquitin chains, the ubiquitin targets of hFBH1 are still unknown (22).
The aim of our study was to identify a human orthologue for budding yeast Srs2. Here we show that the F-box DNA helicase hFBH1 suppresses specific recombination defects of S. cerevisiae srs2 mutants. Furthermore, when hFBH1 substitutes for Srs2, the PRR functions required to induce the K164 ubiquitylation of PCNA are dispensable for cell survival upon DNA damage treatment. The F-box domain is essential for hFBH1 functions in substituting for Srs2 and PRR roles, and it controls the DNA damage-induced turnover of hFBH1 by cooperating with yeast SCF components. Overall, our data indicate that hFBH1 represents a bona fide functional orthologue of budding yeast Srs2 that, in addition, during its evolution might have acquired self-regulatory properties necessary to modulate its DNA recombination functions.
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MATERIALS AND METHODS |
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N and hfbh1-hd strains were constructed using the same strategy by amplifying a PCR fragment lacking the F-box motif or a PCR fragment containing a K520A mutation obtained by site-direct mutagenesis of plasmid pIC1-HIS3. The F-SRS2 strain was constructed by inserting the hFBH1 F-box domain into the NotI site of the pG35 plasmid (28) to generate the pSAPO1 plasmid. PstI-linearized pSAPO1 plasmid was used to integrate the F-SRS2 construct at the SRS2 locus.
Standard genetic analyses were performed according to published procedures (42). An intrachromosomal recombination assay was performed using yeast strains carrying a heteroallelic duplication of LEU2, with URA3 between the LEU2 genes, as previously described (25). Spot assays were performed by evaluating cellular growth on synthetic complete medium containing adenine at a final concentration of 0.7 g/liter, with or without methyl methanesulfonate (MMS), hydroxyurea (HU), and 4-nitroquinolone-1-oxide (4-NQO; Sigma). All spot tests were repeated at least three times in independent experiments, using different yeast transformants or segregants to ensure the reproducibility of the results. Dose-response killing curves for zeocin (Invitrogen) were determined by plating serial dilutions of exponentially growing cells treated for 1 hour with or without the mutagen; to measure UV light sensitivity, a known number of cells were plated from exponentially growing cultures and exposed to different UV doses. CFU were evaluated after 3 to 5 days at 28°C. All survival curves were repeated three times in independent experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot procedures were done as previously described (28), using anti-Myc 9E11 (Bio Optica, United Kingdom) and 
-tubulin (Oxford Biotechnology, United Kingdom) antibodies. Nocodazole and cycloheximide (Sigma) were used at final concentrations of 10 µg/ml and 50 µg/ml, respectively. The protein quantification analysis shown in Fig. 5 was carried out using Image J (NIH) software. The relative amounts of the hFBH1 protein were normalized by using tubulin as a loading control, as calculated in independent experiments.
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mutants. For this analysis, we replaced the SRS2 open reading frame at its chromosomal location with the human cDNA encoding the hFBH1 protein. In the resulting srs2::hFBH1 strain, the yeast SRS2 promoter, which is responsible for the cell cycle and the DNA damage-induced regulation of the original transcript (16), controls the expression of the hFBH1 cDNA insert. By using Western blot analysis, we found that the hFBH1 protein is expressed in the yeast strain, although its level is lower than the level of the endogenous Srs2 protein (see Fig. 3C). srs2 mutants are characterized by hyperactivation of spontaneous recombination (25, 41), and such a deletion is lethal in the absence of RAD54 (23) or SGS1 (14), which are both required for the maturation of recombination intermediates. The cell lethality of srs2 rad54 and srs2 sgs1 double mutants is suppressed by deleting RAD51 (14, 23, 44), consistent with the idea that Srs2 dismantles Rad51 nucleofilaments (26, 52). Using a genetic system that allows the analysis of gene conversion and recombination-mediated deletion events occurring between directed repeats, we confirmed previous observations (25) indicating that in srs2 mutants the rate of gene conversion is enhanced four to five times compared to that of wt cells. We found that the gene conversion rate was reduced in the srs2::hFBH1 strain compared to that of the srs2 strain (Fig. 2A), indicating that hFBH1 is able to counteract recombination in the absence of Srs2. We also found, by tetrad analysis, that while srs2 mutants are synthetically lethal in combination with rad54 deletion, srs2::hFBH1 rad54 double mutants are viable (Fig. 2B). We then tested the ability of hFBH1 to prevent cell lethality in srs2 sgs1 double mutants and found that the presence of a wt copy of SRS2, carried by a URA3 plasmid and counterselectable by treatment with 5-fluoroorotic acid, is much less critical for cell survival in srs2::hFBH1 sgs1 mutants than in srs2 sgs1 mutants (Fig. 2C). srs2, but not sgs1, mutants require RAD27 for cell viability (9, 23). RAD27 encodes a Flap endonuclease implicated in processing of Okazaki fragments during DNA replication, and rad27 mutants also exhibit synthetic lethality in combination with HR mutants (49). These genetic observations might imply that the DNA lesions generated by the absence of Rad27 are repaired mainly through HR and are not in contrast with the possibility that Srs2 favors recombinational repair in this context. rad27 cells were crossed with srs2 or srs2::hFBH1 cells, and tetrad analysis revealed that while srs2 rad27 double mutants are synthetically lethal, srs2::hFBH1 rad27 cells are viable (Fig. 2D). We therefore concluded that hFBH1 can also substitute for Srs2 in processing DNA lesions that spontaneously arise in rad27 mutants. Taken together, the data in Fig. 2 indicate that hFBH1 suppresses the spontaneous recombination defects occurring in the absence of a functional Srs2 protein.
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cells to different mutagens. As shown in Fig. 3A, we found that hFBH1 suppresses the hypersensitivity of srs2 mutants to MMS, HU, and 4-NQO. Similarly, hFBH1 suppresses srs2 hypersensitivity to UV light and zeocin, as assessed by treating exponentially growing cells with increasing doses of mutagens (Fig. 3B).
We then dissected the contributions of the helicase and F-box domains to hFBH1 function by creating two specific mutants (Fig. 3C) in which the F-box motif was deleted (hfbh1-N) or in which the conserved K within the ATP binding domain, essential for the enzymatic activities of the yeast Srs2 protein (25), was changed to A (hfbh1-hd). Both hfbh1 alleles were replaced at the SRS2 locus, and using Western blot analysis, we found that the mutated proteins were expressed at least at the same level as wt hFBH1, both under untreated conditions and in response to MMS (Fig. 3C). srs2::hfbh1-N and srs2::hfbh1-hd strains showed increased sensitivity to drug treatments, resembling that of srs2 mutants, compared to the srs2::hFBH1 strain (Fig. 3A). Hence, both the helicase and the ubiquitin ligase functions of hFBH1 are essential for suppressing the DNA damage sensitivity of srs2 mutants. Moreover, the DNA damage sensitivities of rad51, srs2 rad51, and srs2::hFBH1 rad51 strains were comparable, indicating that hFBH1 suppresses srs2 DNA damage sensitivity only in the presence of a functional HR pathway (Fig. 3A).
It is thought that Srs2 channels DNA lesions into the PRR pathway by preventing HR (5, 45). According to this model, the elevated DNA damage sensitivities of PRR-deficient mutants can be rescued partially by deleting SRS2 in the presence of a functional HR pathway (2, 6, 13, 41, 43, 50). We therefore tested whether hFBH1 in the absence of Srs2 could restore the MMS sensitivity of PRR-deficient mutants. As previously shown, we found that the deletion of SRS2 partially rescued the MMS sensitivities of rad5, rad18, rad6, and ubc13 mutants (Fig. 4A); surprisingly, we found that rad5 srs2::hFBH1, rad18 srs2::hFBH1, and ubc13 srs2::hFBH1 mutants were more resistant to MMS treatment than rad5 srs2, rad18 srs2, and ubc13 srs2 mutants, while rad6 srs2::hFBH1 cells were more sensitive to MMS than rad6 srs2 mutants (Fig. 4A). In response to DNA damage, the PRR factors deleted in the above mutants induce the mono- and polyubiquitylation of PCNA at the conserved K164 residue; srs2 mutants rescue the DNA damage sensitivity of ubiquitylation-defective pcnaK164R mutants (18). We found that hFBH1 also rescues the MMS sensitivity of pcnaK164R mutants in the absence of Srs2 and, similar to what was found for rad5, rad18, and ubc13 mutants, that this rescue is better than that by deletion of SRS2 itself, although not to the wt level (Fig. 4A). Thus, ubiquitylation of PCNA at K164 is also dispensable for MMS survival when hFBH1 substitutes for Srs2.
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N did not rescue the MMS sensitivity of rad5 mutants (Fig. 4B). Notably, we found that hfbh1-hd mutants behaved similarly to hfbh1-N mutants (Fig. 4B), suggesting that a functional hFBH1 helicase domain is also required for rad5 suppression. Since we cannot rule out that deletion of the F-box domain partially affects the helicase activity of hFBH1 (22), we tested the contribution of the F-box domain to the suppression of MMS sensitivity of PRR-deficient mutants in a different way. We asked if the addition of the hFBH1 F-box domain to the yeast Srs2 protein could be sufficient to relieve the need for PRR factors. To test this hypothesis, we created a hybrid SRS2 gene carrying the coding region for the F-box domain of hFBH1 (Fig. 3C) and used this chimera to replace endogenous SRS2 in PRR-deficient backgrounds. We found that the F-box-Srs2 protein (F-Srs2) restored the MMS sensitivity of rad5 or rad18 cells to a similar extent to that by hFBH1 (Fig. 4B). Hence, the hybrid F-box Srs2 protein recapitulates hFBH1 properties in suppressing PRR mutants.
We noted that the MMS sensitivity of rad5 srs2::hFBH1rad51 cells was identical to that of rad5 srs2 rad51 or rad5 rad51 cells, indicating that the suppression of the DNA damage sensitivity of rad5 mutants by hFBH1 requires a functional RAD51 gene (Fig. 4B). Conversely, neither the inactivation of RAD30 nor that of REV3, resulting in ablation of the translesion DNA polymerase 
and DNA polymerase 
, respectively, affected the capability of hFBH1 to suppress the MMS sensitivity of rad5 mutants in the absence of SRS2 (data not shown).
Altogether, the data presented in Fig. 4 indicate that the replacement of Srs2 with hFBH1 suppresses PRR mutant repair defects in the presence of an intact HR pathway and that the F-box domain, in association with functional UvrD helicase activity, plays an essential role in this suppression.
In the course of our studies, we noticed that the deletion of the F-box domain in hfbh1-N mutants led to an increase of the hFBH1 protein level, while its addition to endogenous Srs2 in F-SRS2 strains correlated with a reduced Srs2 protein level (Fig. 3C). In order to better characterize this aspect, we measured hFBH1 protein stability in yeast strains, using the protein synthesis inhibitor cycloheximide.
Different cultures of a yeast strain carrying a Myc-tagged version of hFBH1 were treated with increasing doses of MMS and then exposed to cycloheximide to prevent further protein synthesis. As shown in Fig. 5A, the hFBH1 protein level abruptly decreased in time upon protein synthesis inhibition, and in particular, the hFBH1 turnover rate increased by raising the dose of MMS, as also judged by quantification analysis. Hence, although our analysis suggests that hFBH1 is subjected to rapid degradation even under unperturbed conditions (data not shown), the DNA damage treatment further stimulated its turnover. In contrast, the turnover of a truncated version of hFBH1 lacking the F-box motif was dramatically impaired in MMS-treated cells (Fig. 5B). Given that hFBH1 interacts with Skp1 through the F-box domain to form an SCF complex in vitro (22), we asked if yeast SCF factors could be required for hFBH1 protein turnover under our experimental conditions. As shown in Fig. 5B, we found that hFBH1 degradation is indeed prevented by inactivating the Cdc53 or Skp1 SCF component by using temperature-sensitive alleles.
Overall, these findings indicate that hFBH1 is subject to rapid turnover, which is further stimulated by DNA damage treatment, and that this turnover depends on a functional F-box domain and yeast SCF-dependent degradation machinery.
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DISCUSSION |
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hFBH1 is highly similar to Srs2 within the helicase domain, but it also carries an F-box motif that self-regulates its turnover.
With the aim of searching for putative human orthologues of S. cerevisiae Srs2, we compared by BLAST searching just its conserved UvrD helicase domain with the H. sapiens genome and identified the F-box DNA helicase hFBH1 as the protein with the best score. Indeed, within the helicase domain, hFBH1 shares a high degree of similarity with all members of the UvrD protein family, including S. pombe Srs2 and E. coli UvrD. Beyond the helicase domain, hFBH1 shares poor homology with UvrD proteins, and it exclusively contains an F-box domain. Bona fide orthologues of hFBH1 have been identified in S. pombe, mouse, and chicken cells (21, 24). Considering the similarities and differences between Srs2 and Fbh1 protein subgroups, they likely constitute a single protein family, resembling the case of RecQ helicases, where the different members share homology mainly within their helicase domains (17). Notably, for S. pombe, members of the two subgroups have been described, and genetic data suggest that fbh1 srs2 double mutants are lethal due to unrestrained HR (32, 33). In particular, it has been suggested that S. pombe Fbh1 has a role in disassembling Rad51 nucleofilaments, either those formed in the absence of HR mediator proteins (33) or those created after strand invasion (32). Similarly, in DT40 chicken cells, Fbh1, by cooperating with the BLM RecQ helicase, acts in limiting SCEs (24). Thus, in different organisms, Fbh1 functions rely on recombination, and our findings indicate that hFBH1 can functionally substitute for budding yeast Srs2.
As mentioned above, hFBH1 has an F-box domain, a feature not shared with S. cerevisiae Srs2. The F-box proteins are the interchangeable recognition subunits of ubiquitin ligase SCF complexes that control protein degradation in different cellular processes (54). In vitro studies indeed indicate that hFBH1 is part of an SCF complex that promotes the formation of ubiquitin chains, although the targets modified in vivo are not known (22). Consistent with findings obtained by studying the S. pombe Fbh1 counterpart (32, 33), our data indicate that the F-box domain is necessary for hFBH1 function(s) in substituting for Srs2, and in addition, they raise the possibility that one target of the F-box domain is hFBH1 itself. Indeed, based on data from yeast, hFBH1 is an unstable protein whose turnover is further stimulated by DNA damage and depends on a functional F-box domain and SCF components.
hFBH1 fulfils S. cerevisiae Srs2 functions in HR. Despite the presence of the F-box motif, does hFBH1 accomplish the specific Srs2 recombination functions? In fact, our data indicate that hFBH1 suppresses many srs2-specific recombination defects, thus indicating that hFBH1 is able to substitute for Srs2 in processing recombination intermediates in different contexts. In particular, it has been suggested that certain srs2 phenotypes, including synthetic lethality with rad54 or sgs1 mutants and UV hypersensitivity, can be ascribed to the accumulation of toxic Rad51 nucleofilaments (1, 14, 23), a hypothesis sustained by the finding that Srs2 displaces Rad51 nucleofilaments in vitro (26, 52). It is not known whether Fbh1 proteins have such DNA translocase activity, but since they are members of the UvrD family, it would not be surprising. In addition, Srs2 is also required for DSB repair by HR (3, 19, 20, 35), a role that might also explain the hypersensitivity of srs2 mutants to DNA-damaging agents that cause DSBs or their synthetic lethality with rad27 mutants. In these contexts, Srs2 might act in favoring recombinational repair. It was suggested that the prorecombinational role of Srs2 still reflects its ability to dislodge aberrant or unscheduled Rad51 nucleofilaments that might prevent recombinational repair (1, 33). Otherwise, Srs2 and Fbh1 might also counteract Rad51 nucleofilaments after strand invasion, thus constraining SCE events to the level necessary to repair interrupted DNA molecules. Notably, a role for S. cerevisiae Srs2 and even for S. pombe Fbh1 downstream of the Rad51 strand invasion step is supported by genetic findings (20, 32, 39). Finally, since both Srs2 and Fbh1 unwind the DNA duplex in vitro as proper DNA helicases (21, 40), they might have a more direct role in vivo in promoting recombination by extending the D loop once strand invasion has occurred (35).
hFBH1 faces error-free PRR functions by the aim of its F box.
What is more surprising in our findings is that hFBH1, in the absence of SRS2, suppresses the DNA damage sensitivity of PRR mutants, while in contrast, Srs2 causes cell death of PRR mutants (5, 45). As a consequence, hFBH1 and Srs2 have apparently opposite effects on the recombination outcome in this context: while Srs2 is thought to kill PRR mutants by preventing recombination, hFBH1 might rescue PRR mutants from cell lethality by favoring recombination. The requirement of HR for this suppression might support this possibility. Notably, the suppression of PRR mutant cell lethality by hFBH1 cannot be ascribed to the observation that hFBH1 might be present in limiting amounts with respect to Srs2. In fact, hFBH1 suppresses the MMS sensitivity of PRR mutants even more efficiently than that of srs2 mutants. Our data rather suggest that this suppression entails an active UvrD helicase whose turnover depends on its F-box motif, resulting in an enzyme that efficiently favors recombinational repair.
According to the current model, Srs2 exerts antirecombination functions when it is recruited by sumoylated PCNA (34, 36). The association of Srs2 with sumoylated PCNA depends on its C-terminal tail, and its removal induces a PRR suppression phenotype resembling the one caused by preventing PCNA sumoylation (36). Although the C-terminal domain of Srs2 has not been conserved in hFBH1, our data indicate that the hybrid F-Srs2 protein that retains the SUMO-PCNA binding domain (Fig. 3C) is still able to suppress the MMS sensitivity of PRR mutants. Furthermore, both F-Srs2 and hFBH1 suppress PRR mutants better than mutations that prevent the sumoylation of PCNA, whose phenotypes in this regard resemble those of srs2 mutants (36). Thus, the presence of the F-box domain rather than the absence of the C-terminal tail seems to be crucial for the suppression of PRR mutants by hFBH1 or F-Srs2.
Although other possibilities could be envisaged, we favor one interpretation of our data that might suggest a novel functional relationship between Srs2 and PRR factors in S. cerevisiae, as follows. In response to DNA damage, Srs2 might act mainly in favoring recombinational repair and PRR factors might enhance this Srs2 prorecombinational activity. In PRR mutants, Srs2 becomes toxic because it is unable to stimulate proper recombination events, and it simultaneously prevents access to DNA lesions of other factors that promote HR in its absence. In this scenario, hFBH1, by the aim of its F-box, fulfils Srs2 functions in HR and the PRR regulatory role in Srs2 activity at the same time.
But how could PRR genes influence Srs2 duties, and what is the mechanism by which the F-box hFBH1 helicase could substitute for PRR functions? Studies with human cells showed that hFBH1 is a component of the SCF ubiquitin ligase complex (22), and SCF complexes have been implicated in K48 ubiquitylation and in 26S proteasome-dependent degradation of their targets (54). Therefore, one likely possibility is that hFBH1 disruption occurs through an autoubiquitylation mechanism (Fig. 6). Since the F-box domain self-regulates the turnover of hFBH1 and is required for the suppression of PRR mutants, one interesting hypothesis is that the control exerted by the PRR pathway on Srs2 relies on a similar mechanism. Rad6/Rad18 and Rad5/Ubc13 cooperate to assemble ubiquitin via K63 chains, which is not a typical signal for protein degradation (37); thus, it is unlikely that the PRR pathway would directly promote Srs2 disruption. However, in response to DNA damage, the PRR pathway promotes PCNA ubiquitylation (34, 36). Given that hFBH1 also rescues ubiquitin-defective pcnaK164R mutants, an intriguing possibility is that one of the essential functions of PRR factors, by inducing PCNA modification, is in turn to influence the stability of Srs2 (Fig. 6). This hypothesis might not be entirely surprising, since it was recently shown that the replication factor Cdt1 is ubiquitylated and degraded, also in response to DNA damage, once it is recruited by PCNA to the DNA template (15). In light of this, we speculate that Srs2 degradation in its natural context is promoted by an SCF complex and that an unidentified endogenous F-box protein in yeast could perform the same F-box-dependent function of hFBH1 (Fig. 6). Therefore, the PRR pathway, by inducing the modification of PCNA, might at the same time influence the capability of an SCF complex to disrupt Srs2 and possibly other factors bound to DNA, maybe by stimulating the recruitment of a putative F-box protein. In this view, hFBH1 does not require certain PRR functions to survive DNA damage because it is able to self-regulate its turnover. Similarly, the proper turnover of endogenous Srs2 in PRR mutants can be reestablished by adding the sole F-box domain of hFBH1 to the helicase.
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Concluding remarks. Genetic analysis performed with fission yeast suggests that the two UvrD helicases Fbh1 and Srs2 have nonredundant functions in recombination (32, 33). An intriguing possibility is that the recombination role(s) performed by the sole Srs2 helicase in budding yeast, regulated by the PRR pathway and possibly by phosphorylation (28), is achieved by distinct polypeptides in fission yeast. Notably, S. cerevisiae Srs2, exclusively among UvrD helicases, possesses a long C-terminal tail (Fig. 1B) containing most of the DNA damage-inducible phosphosites (8), whose deletion alone is sufficient to suppress PRR mutants and which is required for the physical interactions with both sumoylated PCNA (36) and Rad51 (26). If the functions of S. pombe Srs2 and Fbh1 could be reassumed by a sole PRR-regulated UvrD helicase in S. cerevisiae, then this would explain why the genetic relationships between SRS2 and PRR genes in S. pombe, or even in N. crassa, are less consistent with those depicted in S. cerevisiae. In fact, while budding yeast srs2 mutants suppress the DNA damage sensitivity of PRR mutants, S. pombe or N. crassa srs2 mutants do not (10, 48). Furthermore, the same hypothesis might justify why the timing of ubiquitylation- and sumoylation-dependent modifications of PCNA, which are crucial for the regulation of Srs2 recombination functions in S. cerevisiae (34, 36), has not been conserved equally in S. pombe (12). It is interesting that a phylogenetic analysis performed on hemiascomycete yeasts revealed that the SRS2 locus is duplicated in tandem in certain species, and it is thought that gene duplication might facilitate their rapid evolution toward specialized functions (38).
In conclusion, our data are compatible with the idea that hFBH1 is a bona fide human orthologue of S. cerevisiae Srs2 that, thanks to F-box-dependent functions, has evolved to execute its recombination role(s) independently of the PCNA modification status, as instead happens in budding yeast.
It will be a challenge for the future to establish whether other proteins functionally related to hFBH1 are present in human cells and whether inactivation of hFBH1 might have important implications for human health, like the case for the RecQ helicases BLM, WRN, and RECQ4 (17).
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro and by the European Community. I.C. is a recipient of an AIRC Unicredito Italiano fellowship.
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FOOTNOTES |
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Published ahead of print on 27 August 2007.
Present address: Department of Genome and Computational Biology, Lawrence Berkeley National Laboratory, MS 84-171, 1 Cyclotron Road, Berkeley, CA 94720.
Present address: Banting and Best Department of Medical Research and Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada.
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REFERENCES |
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2. Aboussekhra, A., R. Chanet, Z. Zgaga, C. Cassier-Chauvat, M. Heude, and F. Fabre. 1989. RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene. Nucleic Acids Res. 17: 7211-7219.
3. Aylon, Y., B. Liefshitz, G. Bitan-Banin, and M. Kupiec. 2003. Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 23: 1403-1417.
4. Branzei, D., J. Sollier, G. Liberi, X. Zhao, D. Maeda, M. Seki, T. Enomoto, K. Ohta, and M. Foiani. 2006. Ubc9- and mms21-mediated sumoylation counteracts recombinogenic events at damaged replication forks. Cell 127: 509-522.[CrossRef][Medline]
5. Broomfield, S., T. Hryciw, and W. Xiao. 2001. DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. Mutat. Res. 486: 167-184.[Medline]
6. Broomfield, S., and W. Xiao. 2002. Suppression of genetic defects within the RAD6 pathway by srs2 is specific for error-free post-replication repair but not for damage-induced mutagenesis. Nucleic Acids Res. 30: 732-739.
7. Chanet, R., M. Heude, A. Adjiri, L. Maloisel, and F. Fabre. 1996. Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase. Mol. Cell. Biol. 16: 4782-4789.[Abstract]
8. Chiolo, I., W. Carotenuto, G. Maffioletti, J. H. Petrini, M. Foiani, and G. Liberi. 2005. Srs2 and Sgs1 DNA helicases associate with Mre11 in different subcomplexes following checkpoint activation and CDK1-mediated Srs2 phosphorylation. Mol. Cell. Biol. 25: 5738-5751.
9. Debrauwere, H., S. Loeillet, W. Lin, J. Lopes, and A. Nicolas. 2001. Links between replication and recombination in Saccharomyces cerevisiae: a hypersensitive requirement for homologous recombination in the absence of Rad27 activity. Proc. Natl. Acad. Sci. USA 98: 8263-8269.
10. Doe, C. L., and M. C. Whitby. 2004. The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast. Nucleic Acids Res. 32: 1480-1491.
11. Fabre, F., A. Chan, W. D. Heyer, and S. Gangloff. 2002. Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proc. Natl. Acad. Sci. USA 99: 16887-16892.
12. Frampton, J., A. Irmisch, C. M. Green, A. Neiss, M. Trickey, H. D. Ulrich, K. Furuya, F. Z. Watts, A. M. Carr, and A. R. Lehmann. 2006. Postreplication repair and PCNA modification in Schizosaccharomyces pombe. Mol. Biol. Cell 17: 2976-2985.
13. Friedl, A. A., B. Liefshitz, R. Steinlauf, and M. Kupiec. 2001. Deletion of the SRS2 gene suppresses elevated recombination and DNA damage sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae. Mutat. Res. 486: 137-146.[Medline]
14. Gangloff, S., C. Soustelle, and F. Fabre. 2000. Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases. Nat. Genet. 25: 192-194.[CrossRef][Medline]
15. Green, C. M. 2006. One ring to rule them all? Another cellular responsibility for PCNA. Trends Mol. Med. 12: 455-458.[CrossRef][Medline]
16. Heude, M., R. Chanet, and F. Fabre. 1995. Regulation of the Saccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle, meiosis and after irradiation. Mol. Gen. Genet. 248: 59-68.[CrossRef][Medline]
17. Hickson, I. D. 2003. RecQ helicases: caretakers of the genome. Nat. Rev. Cancer 3: 169-178.[CrossRef][Medline]
18. Hoege, C., B. Pfander, G. L. Moldovan, G. Pyrowolakis, and S. Jentsch. 2002. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419: 135-141.[CrossRef][Medline]
19. Ira, G., and J. E. Haber. 2002. Characterization of RAD51-independent break-induced replication that acts preferentially with short homologous sequences. Mol. Cell. Biol. 22: 6384-6392.
20. Ira, G., A. Malkova, G. Liberi, M. Foiani, and J. E. Haber. 2003. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115: 401-411.[CrossRef][Medline]
21. Kim, J., J. H. Kim, S. H. Lee, D. H. Kim, H. Y. Kang, S. H. Bae, Z. Q. Pan, and Y. S. Seo. 2002. The novel human DNA helicase hFBH1 is an F-box protein. J. Biol. Chem. 277: 24530-24537.
22. Kim, J. H., J. Kim, D. H. Kim, G. H. Ryu, S. H. Bae, and Y. S. Seo. 2004. SCFhFBH1 can act as helicase and E3 ubiquitin ligase. Nucleic Acids Res. 32: 2287-2297.
23. Klein, H. L. 2001. Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Delta with other DNA repair genes in Saccharomyces cerevisiae. Genetics 157: 557-565.
24. Kohzaki, M., A. Hatanaka, E. Sonoda, M. Yamazoe, K. Kikuchi, N. Vu Trung, D. Szuts, J. E. Sale, H. Shinagawa, M. Watanabe, and S. Takeda. 2007. Cooperative roles of vertebrate Fbh1 and Blm DNA helicases in avoidance of crossovers during recombination initiated by replication fork collapse. Mol. Cell. Biol. 27: 2812-2820.
25. Krejci, L., M. Macris, Y. Li, S. Van Komen, J. Villemain, T. Ellenberger, H. Klein, and P. Sung. 2004. Role of ATP hydrolysis in the antirecombinase function of Saccharomyces cerevisiae Srs2 protein. J. Biol. Chem. 279: 23193-23199.
26. Krejci, L., S. Van Komen, Y. Li, J. Villemain, M. S. Reddy, H. Klein, T. Ellenberger, and P. Sung. 2003. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423: 305-309.[CrossRef][Medline]
27. Lawrence, C. W. 2007. Following the RAD6 pathway. DNA Repair (Amsterdam) 6: 676-686.[CrossRef]
28. Liberi, G., I. Chiolo, A. Pellicioli, M. Lopes, P. Plevani, M. Muzi-Falconi, and M. Foiani. 2000. Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity. EMBO J. 19: 5027-5038.[CrossRef][Medline]
29. Liberi, G., G. Maffioletti, C. Lucca, I. Chiolo, A. Baryshnikova, C. Cotta-Ramusino, M. Lopes, A. Pellicioli, J. E. Haber, and M. Foiani. 2005. Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes Dev. 19: 339-350.
30. Maftahi, M., J. C. Hope, L. Delgado-Cruzata, C. S. Han, and G. A. Freyer. 2002. The severe slow growth of Deltasrs2 Deltarqh1 in Schizosaccharomyces pombe is suppressed by loss of recombination and checkpoint genes. Nucleic Acids Res. 30: 4781-4792.
31. Martin, V., C. Chahwan, H. Gao, V. Blais, J. Wohlschlegel, J. R. Yates III, C. H. McGowan, and P. Russell. 2006. Sws1 is a conserved regulator of homologous recombination in eukaryotic cells. EMBO J. 25: 2564-2574.[CrossRef][Medline]
32. Morishita, T., F. Furukawa, C. Sakaguchi, T. Toda, A. M. Carr, H. Iwasaki, and H. Shinagawa. 2005. Role of the Schizosaccharomyces pombe F-box DNA helicase in processing recombination intermediates. Mol. Cell. Biol. 25: 8074-8083.
33. Osman, F., J. Dixon, A. R. Barr, and M. C. Whitby. 2005. The F-box DNA helicase Fbh1 prevents Rhp51-dependent recombination without mediator proteins. Mol. Cell. Biol. 25: 8084-8096.
34. Papouli, E., S. Chen, A. A. Davies, D. Huttner, L. Krejci, P. Sung, and H. D. Ulrich. 2005. Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol. Cell 19: 123-133.[CrossRef][Medline]
35. Paques, F., and J. E. Haber. 1997. Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae. Mol. Cell. Biol. 17: 6765-6771.[Abstract]
36. Pfander, B., G. L. Moldovan, M. Sacher, C. Hoege, and S. Jentsch. 2005. SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436: 428-433.[Medline]
37. Pickart, C. M., and D. Fushman. 2004. Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8: 610-616.[CrossRef][Medline]
38. Richard, G. F., A. Kerrest, I. Lafontaine, and B. Dujon. 2005. Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination. Mol. Biol. Evol. 22: 1011-1023.
39. Robert, T., D. Dervins, F. Fabre, and S. Gangloff. 2006. Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover. EMBO J. 25: 2837-2846.[CrossRef][Medline]
40. Rong, L., and H. L. Klein. 1993. Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae. J. Biol. Chem. 268: 1252-1259.
41. Rong, L., F. Palladino, A. Aguilera, and H. L. Klein. 1991. The hyper-gene conversion hpr5-1 mutation of Saccharomyces cerevisiae is an allele of the SRS2/RADH gene. Genetics 127: 75-85.[Abstract]
42. Rose, M., F. Winston, and P. Hieter. 1990. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
43. Schiestl, R. H., S. Prakash, and L. Prakash. 1990. The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway. Genetics 124: 817-831.[Abstract]
44. Schild, D. 1995. Suppression of a new allele of the yeast RAD52 gene by overexpression of RAD51, mutations in srs2 and ccr4, or mating-type heterozygosity. Genetics 140: 115-127.[Abstract]
45. Smirnova, M., and H. L. Klein. 2003. Role of the error-free damage bypass postreplication repair pathway in the maintenance of genomic stability. Mutat. Res. 532: 117-135.[Medline]
46. Stelter, P., and H. D. Ulrich. 2003. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425: 188-191.[CrossRef][Medline]
47. Sung, P., and H. Klein. 2006. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat. Rev. Mol. Cell. Biol. 7: 739-750.[CrossRef][Medline]
48. Suzuki, K., A. Kato, Y. Sakuraba, and H. Inoue. 2005. Srs2 and RecQ homologs cooperate in mei-3-mediated homologous recombination repair of Neurospora crassa. Nucleic Acids Res. 33: 1848-1858.
49. Symington, L. S. 1998. Homologous recombination is required for the viability of rad27 mutants. Nucleic Acids Res. 26: 5589-5595.
50. Ulrich, H. D. 2001. The srs2 suppressor of UV sensitivity acts specifically on the RAD5- and MMS2-dependent branch of the RAD6 pathway. Nucleic Acids Res. 29: 3487-3494.
51. Veaute, X., S. Delmas, M. Selva, J. Jeusset, E. Le Cam, I. Matic, F. Fabre, and M. A. Petit. 2005. UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli. EMBO J. 24: 180-189.[CrossRef][Medline]
52. Veaute, X., J. Jeusset, C. Soustelle, S. C. Kowalczykowski, E. Le Cam, and F. Fabre. 2003. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423: 309-312.[CrossRef][Medline]
53. Wang, S. W., A. Goodwin, I. D. Hickson, and C. J. Norbury. 2001. Involvement of Schizosaccharomyces pombe Srs2 in cellular responses to DNA damage. Nucleic Acids Res. 29: 2963-2972.
54. Willems, A. R., M. Schwab, and M. Tyers. 2004. A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin. Biochim. Biophys. Acta 1695: 133-170.[Medline]
55. Wu, L., and I. D. Hickson. 2006. DNA helicases required for homologous recombination and repair of damaged replication forks. Annu. Rev. Genet. 40: 279-306.[CrossRef][Medline]
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